The present invention relates to hormone receptor proteins and genes encoding them, modification of such receptors and genes by recombinant DNA and other genetic engineering techniques, plus uses of such receptors and genes, both unmodified and modified. More particularly, the invention concerns steroid and thyroid hormone receptors and associated genes. Most particularly, it concerns human glucocorticoid, mineralocorticoid and thyroid hormone receptors and genes for them. In addition the invention relates to a novel bioassay system for determining the functionality of hormone receptor proteins coded for by receptor DNA clones, plus novel methods for inducing and controlling expression of genes whose transcription is activated by hormones complexed with receptor proteins.
Transcriptional regulation of development and homeostasis in complex eukaryotes, including humans and other mammals, birds, and fish, is controlled by a wide variety of regulatory substances, including steroid and thyroid hormones. These hormones exert potent effects on development and differentiation in phylogenetically diverse organisms and their actions are mediated as a consequence of their interactions with specific, high affinity binding proteins referred to as receptors. See generally, Jensen, et al., (1972); Gorski, et al., (1976); Yamamoto, et al., (1976); O""Malley, et al., (1969); Hayward, et al., (1982); and Asburner, et al., (1978).
Receptor proteins, each especially specific for one of the several classes of cognate steroid hormones (i.e., estrogens (estrogen receptor), progestogens (progesterone receptor), glucocorticoids (glucocorticoid receptor), androgens (androgen receptor), aldosterones (mineralocorticoid receptor) or for cognate thyroid hormones (thyroid hormone receptor), are known and distributed in a tissue specific fashion. See Horwitz, et al., (1978) and Pamiter, et al., (1976).
Turning now to the interaction of hormones and receptors, it is known that a steroid or thyroid hormone enters cells by facilitated diffusion and binds to its specific receptor protein, initiating an alosteric alteration of the protein. As a result of this alteration, the hormone/receptor complex is capable of binding to certain specific sites on chromatin with high affinity. See Yamamoto, et al., (1972) and Jensen, et al., (1968).
It is also known that many of the primary effects of steroid and thyroid hormones involve increased transcription of a subset of genes in specific cell types. See Peterkofsky, et al., (1968) and McKnight, et al., (1968). Moreover, there is evidence that activation of transcription (and, consequently, increased expression) of genes which are responsive to steroid and thyroid hormones (through interaction of chromatin with hormone receptor/hormone complex) is effected through binding of the complex to enhancers associated with the genes. (See Khoury, et al., 1983.)
In any case, a number of steroid hormone and thyroid hormone responsive transcriptional control units, some of which have been shown to include enhancers, have been identified. These include the mouse mammary tumor virus 5xe2x80x2-long terminal repeat (MTV LTR), responsive to glucocorticoid, aldosterone and androgen hormones; the transcriptional control units for mammalian growth hormone genes, responsive to glucocorticoids, estrogens, and thyroid hormones; the transcriptional control units for mammalian prolactin genes and progesterone receptor genes, responsive to estrogens; the transcriptional control units for avian ovalbumin genes, responsive to progesterones; mammalian metallothionein gene transcriptional control units, responsive to glucocorticoids; and mammalian hepatic alpha2u-globulin gene transcriptional control units, responsive to androgens, estrogens, thyroid hormones and glucocorticoids. (See the Introduction portion of Experimental Section I of this Specification for references.)
A major obstacle to further understanding and more practical use of the steroid and thyroid hormone receptors has been the lack of availability of the receptor proteins, in sufficient quantity and sufficiently pure form, to allow them to be adequately characterized. The same is true for the DNA gene segments which encode them. Lack of availability of these DNA segments has prevented in vitro manipulation and in vivo expression of the receptor-coding genes, and consequently the knowledge such manipulation and expression will yield.
The present invention is directed to overcoming these problems of short supply of adequately pure receptor material and lack of DNA segments which encode the receptors.
The Background section of the specification refers to the following publications.
1. Asburner, M., and Berendes, H. D. in The Genetics and Biology of Drosophila, Eds. Ashburner, M., and Wright, T. R. F., Vol. 2, pp. 315-395, Academic, London (1978).
2. Gorski, J., and Gannon, F., A. Rev. Physiol., 38:425-450 (1976).
3. Hayward, M. A., Brock, M. L. and Shapiro, D. J., Nucleic Acids Res., 10:8273-8284 (1982).
4. Horwitz, K. B., and McGuire, W. L., J. Biol. Chem., 253:2223-2228 (1978).
5. Jensen, E. V., and DeSombre, E. R., A. Rev. Biochrm., 41:203-230 (1972).
6. Jensen, E. V., et al., Proc. Natl. Acad. Sci. U.S.A., 59:632-638 (1968).
7. Khoury, G., and Gruss, P., Cell, 33:313-314 (1983).
8. McKnight, G. S., and Palmiter, R. D., J. Biol. Chem., 254:9050-9058 (1968).
9. O""Malley, B. W., McGuire, W. L., Kohler, P. O., and Korman, S. G., Recent Prog. Horm. Res., 25:105-160 (1969).
10. Pamiter, R. D., Moore, P. B., Mulvihill, E. R. and Emtage, S., Cell, 8:557-572 (1976).
11. Peterkofsky, B., and Tomkins, G., Proc. Natl. Acad. Sci. U.S.A., 60:222-228 (1968).
12. Yamamoto, K. R., and Alberts, B. M., A. Rev. Biochrm., 45:721-746 (1976).
13. Yamamoto, K. R., and Alberts, B. M., Proc. Natl. Acad. Sci. U.S.A., 69:2105-2109 (1972).
Some of the information disclosed in this specification has been published:
The study disclosed in Experimental Section I has been published as: Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A., Lebo, R., Thompson, E. B., Rosenfeld, M. G., and Evans, R. M., xe2x80x9cPrimary Structure and Expression of a Functional Human Glucocorticoid Receptor cDNAxe2x80x9d, Nature (London), 318:635-641 (December, 1985).
The study disclosed in Experimental Section II has been published as: Giguere, V., Hollenberg, S. M., Rosenfield, M. G., and Evans, R. M., xe2x80x9cFunctional Domains of the Human Glucocorticoid Receptorxe2x80x9d, Cell, 46:645-652 (August, 1986)
The study disclosed in Experimental Section III has been published as: Weinberger, C., Thompson, C. C., Ong, E. S., Lebo, R., Gruol, D. J., and Evans, R. M., xe2x80x9cThe c-erb-A Gene Encodes a Thyroid Hormone Receptorxe2x80x9d, Nature (London), 324:641-646 (December, 1986).
The study disclosed in Experimental Section IV has been published as: Arriza, J. L., Weinberger, C., Cerelli, G., Glaser, T. M., Handelin, B. L., Houseman, D. E., and Evans, R. M., xe2x80x9cCloning of Human Mineralocorticoid Receptor Complementary DNA: Structural and Functional Kinship with the Glucocorticoid Receptorxe2x80x9d, Science, 237:268-275 (July, 1987).
The study disclosed in Experimental Section V is in press as: Giguere, V., Yang, N., Segui, P., and Evans, R. M., xe2x80x9cIdentification of a New Class of Steroid Hormone Receptorsxe2x80x9d.
The study disclosed in Experimental Section VI is in press as: Glass, C. K., Franco, R., Weinberger, C., Albert, V. R., Evans, R. M., and Rosenfeld, M. G., xe2x80x9cA c-erb-A Binding Site in the Rat Growth Hormone Gene Mediates Transactions by Thyroid Hormonexe2x80x9d.
The study disclosed in Experimental Section VII has been published as: Thompson, Catherine C., Weinberger, Cary, Lebo, Roger, and Evans, Ronald M., xe2x80x9cIdentification of a Novel Thyroid Hormone Receptor Expressed in the Mammalian Central Nervous Systemxe2x80x9d, Science, 237:1610-1614 (September, 1987).